The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.
In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.
Metastasis is the final step in the progression of breast cancer 1. It involves a series of steps in which malignant cells are released from the primary tumor and disseminated to other organs 2. The cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system 3. Each step in tumorigenesis is tightly controlled by cells through various genetic and epigenetic changes 4. To study the mechanisms of breast cancer tumorigenesis, mouse model systems have been utilized due to their short lifespan, small size and fast breeding time. Some major advantages of using mouse models in cancer research are the molecular and physiological similarities they share with humans 5.
Established mouse and human cell lines have been used extensively in breast cancer research. Although we can obtain some information about the steps in metastasis using these cells, genetically manipulated animal models with tumor suppressors/oncogenes will give far more information about tumor development and metastasis starting from the embryonic stage. Thus, with these mouse models, we can obtain genetic and developmental regulation of tumor growth. Furthermore, the tumor microenvironment also plays an important role in tumor cell gene expression and it can be difficult to mimic the same scenario with studies using established cell lines. Primary human mammary tumors can also be used to study mouse mammary tumorigenesis. The main disadvantage of using isolated breast tumors is that this newly implanted tumor does not have the mouse’s vasculature and lymphatic tissue. Whereas the genetic mouse model systems for studying mammary tumorigenesis will allow us to monitor the steps of metastasis, analyze the tumor microenvironment, and access to the entire body 5.
The overall goal of this protocol is to study breast tumorigenesis in mice. It is known that breast cancers typically metastasize to the brain, lung and bone. This protocol allows for the isolation of primary mammary tumor cells, as well as, studying breast cancer metastasis to the lung. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. Together, these techniques are useful in studying mouse mammary tumorigenesis.
Dit protocol is voor het isoleren van primaire cellen van een nieuwe muis tumor. Lysaten bereid uit deze werkwijze bruikbaar voor eiwitten, DNA en RNA-analyse. Kankercellen uit de primaire borsttumor metastaseren vaak naar de hersenen, longen en botten 1. Inbegrepen is een techniek om de long voor de detectie van metastasen extraheren. Kleuring van de long met een inktvlek zal voor de visualisatie van tumoren zoals getoond in figuur 2B. Vaststelling van de longen in Z-fix oplossing zal ons to…
The authors have nothing to disclose.
We would like to thank NIH, LACaTS and Ochsner clinic foundation for the financial support.
DMEM | HyClone | SH30243.01 | Store at 4 °C |
0.05% Trypsin-EDTA | Life technologies | 25300-062 | Store at – 20 °C |
Collagenase Type IV | Sigma-Aldrich | C5138 | Store at – 20 °C |
Hyaluronidase | Sigma-Aldrich | H3506 | Store at – 20 °C |
Pen Strep | Life technologies | 15140-122 | Store at 4 °C |
Fetal Bovine Serum (FBS) | Gemini Bio-Products | 100-106 | Store at – 80 °C |
Z-fix | ANATECH | #174 | Store at room temperature |
India Ink | Yasutomo | Store at room temperature | |
AERRANE (Isoflurane) | Baxter | NDC 10019-773-60 | Store at room temperature |
Ethanol | EMD | AX0441-3 | Store at room temperature |